Histologic Analysis of Chronos in an Optogenetic‐Based Auditory Brainstem Implant Model

Abstract

Objectives: Optogenetics affords the potential for improved spatial resolution compared with electric stimulation in future auditory neuroprostheses. No histologic studies have yet examined the virally-mediated gene transfer of Chronos, a new opsin, to the murine cochlear nucleus (CN). Herein, we aim to (1) identify CN regions and neurons receptive to gene transfer of Chronos and (2) describe the morphology of putative cell types that express Chronos. Methods: CBA/CaJ mice underwent CN-targeted injection of Chronos. The Chronos construct consisted of an adeno-associated viral vector (AA2/8), CAG promoter, and a fluorescent marker. Following a 4-week incubation period, mice were sacrificed and intravascularly fixed with paraformaldehyde, and brains were extracted, sucrose cryoprotected, and cryostatically sectioned. Sections of 35-µm thickness were co-labeled with neuron-specific markers microtubule associated protein-2 and anti-tubulin, beta III isoform and DAPI-fluoromounted. Sections of 60-µm thickness were DAPI-fluoromounted, and confocal microscopy revealed cellular morphologies. Results: Opsin-linked fluorescence demonstrates Chronos expression throughout the dorsal CN with contiguous extension routinely into the ventral CN and variably into the auditory nerve and inferior cerebellar peduncle. Chronos localizes to neuronal-specific and nonneuronal populations. Confocal microscopy suggests involvement of a wide array of CN cell types, including morphologies consistent with pyramidal cells and giant cells. Conclusions: Our histologic analyses confirm widespread infection of multiple neuronal populations throughout the CN. This work sets the stage for correlation with ongoing neurophysiology experiments. Future work with CN-specific promoters to target neuronal subpopulations may further improve clinical potential for an optogenetics-based auditory neuroprosthesis.